Communication likely first evolved on our planet as chemical signals between single-cell microorganisms. A fundamental purpose for this type of communication in modern microorganisms is to allow these microbes to self-organize into functional communities. Colonies of the budding yeast, S. cerevisiae, provide an opportunity to investigate this type of community organization due to this model organism's peerless genome annotation and genetic malleability. The Honigberg lab discovered that diploid yeast colonies are organized into a layer of feeder cells underlying a layer of meiotic (sporulating) cells. Furthermore, the relative number of cells and dimensions of the two layers depends on colony environment. Feeder cells may stimulate sporulation in the overall community by providing nutrients to the cells of the overlying layer. In addition to its scientific interest, the health relevance of the proposed research derives from the fact that the spatial organization of pathogenic yeast biofilms contributes significantly to the lethality of hospital-acquired fungal infections. Furthermore, mechanisms of microbial community organization could help elucidate the forces driving organization of tissues and tumors in humans. A long-range goal of the Honigberg lab is to identify the mechanisms that regulate the self- organization of yeast colonies from homogeneous to highly patterned communities. The first specific aim of the application is to test the ?Differential Partitioning provides Environmental Buffer? hypothesis by determining whether the number of feeder cells in colonies correlates with the dependency on these feeder cells for sporulation across a range of conditions. In addition, we will determine whether establishing and maintaining differential partitioning depends on both cell autonomous and cell nonautonomous mechanisms. The second specific aim is to characterize feeder cells with respect to the similarity of expression patterns to quiescent cells found in cultures, and to test the hypotheses that the number of feeder cells in colonies responds to the respiratory state of the colony through mitochondrial signaling. Three complementary approaches are employed to address the above hypotheses. The first approach measures expression of known feeder-cell specific or sporulation-specific genes within intact wild- type or mutant colonies by confocal or multi-photon fluorescent microscopy, and to measure co-localization of these genes in cell populations from resuspended colonies. The second approach examines partitioning and other markers of colony development across a 2-D environmental landscape, i.e. when two environmental conditions (such as temperature or concentration of nutrients) are both varied. The third approach compares gene expression patterns in feeder and sporulation cell layers using FACS-Seq.